The continued discovery of novel genes provides a resource of genetic material for studying the association between genotype and disease. See Kononen, J., et al. (1998) Nat. Med. 4, 844-847. The majority of genetic diseases are due to single base alterations that may be found at multiple sites within one or several genes. See Cooper, D. N., and Krawczak, M. (1990) Hum. Genet. 85, 55-74; Neufeld, E. J. (1998) Hematol. Oncol. Clin. North Am. 12, 1193-1209. For this reason, techniques for nucleic acid analysis often require analysis of multiple loci and sequence variants. For convenience, it is preferable to perform this analysis in a single reaction.
Different colored fluorescent dyes are frequently used to increase the quantity of information obtained from a nucleic acid sample. Mansfield, E. S., et al. (1997) J. Chromatogr. A. 781, 295-305; Samiotaki, M., et al. (1997) Anal. Biochem. 253, 156-161. These dyes can be attached to primers or probes and the products analyzed either during PCR amplification or by post-amplification detection methods. Pritham, G. H., and Wittwer, C. T. (1998) J. Clin. Lig. Assay. 21, 1-9. Although post-amplification analysis increases assay time, post-amplification analysis provides a second level of investigation that multiplies the power of the assay. For example, multicolor fluorescence detection in combination with product sizing has been successful for identity typing using short tandem repeats, genotyping single base changes by minisequencing, genotyping by competitive priming. Mansfield, E. S., et al. (1997) J. Chromatogr. A. 781, 295-305; Pritham, G. H., and Wittwer, C. T. (1998) J. Clin. Lig. Assay. 21, 1-9; Pastinen, T., et al. (1996) Clin. Chem. 42, 1391-1397.
Hybridization probes provide an elegant system for homogenous PCR amplification and genotyping. See Lay, M. J., and Wittwer, C. T. (1997) Clin. Chem. 43, 2262-2267; Bernard, P. S., et al. (1998) Anal. Biochem. 255, 101-107; Bernard, P. S., et al. (1998) Am. J. Pathol. 153, 1055-1061. In one such system, two oligonucleotide probes that hybridize to adjacent regions of a DNA sequence are employed, wherein each oligonucleotide probe is labeled with a respective member of a fluorescent energy transfer pair. In this system, a donor fluorescent dye is excited and transfers energy to an acceptor fluorescent dye if the two dyes are in close proximity, as they would be when hybridized to adjacent regions of the DNA sequence. Post-amplification melting curve analysis allows at least two alleles to be identified using this single pair hybridization probes. This is because variations in DNA sequences create mismatches with the probe resulting in characteristic T.sub.m shifts that are measured by monitoring changes in fluorescence resonance energy transfer during slow heating.
Probes having different T.sub.m s with a variety of alleles can be multiplexed together to increase the power of this technique (Bernard, P. S., et al. (1998) Am. J. Pathol. 153, 1055-1061); however, multiplexing is ultimately limited by the number of T.sub.m s that can be differentiated over a range of probe melting temperatures. The present invention is directed to expanding the power of hybridization probe multiplexing by using color as well as T.sub.m for genotyping different alleles at one time. Multiple probes having different acceptor dyes are employed. Because the acceptor dyes emit at different wavelengths, such multicolor analysis can be used to expand the number of alleles which can be studied at one time.
While multicolor analysis can be used to expand the amount of information obtained in a single melting curve, multicolor analysis requires the use of crosstalk compensation techniques to correct for fluorescence overlap between channels. These methods were originally developed for multiparameter fluorescent monitoring of cells using flow cytometry. Bagwell, C. B., and Adams, E. G. (1993) Ann. N.Y. Acad. Sci. 677, 167-184. While these techniques have proven to be useful, the temperature remains constant in flow cytometry and these methods do not correct for changes in temperature-dependent crossover effects. With melting curve analysis, the temperature ranges from 40 to 95.degree. C., and significant errors can arise if temperature-dependent crossover effects are not corrected. In order to adapt multicolor analysis for use with hybridization probes, algorithm allowances for different gain settings and for temperature effects on fluorescence overlap between channels are necessary.
The present approach is to perform a calibration run during a temperature ramp to define the temperature dependence of each fluorophore. This temperature dependence is then approximated with third degree polynomials. Then, for fluorescent value acquisition during subsequent test runs, the temperature of the acquisition is used to interpolate fluorescence calibration coefficients at that temperature. Without this correction, fluorescent values would be incorrect due to temperature dependent spectral overlap.
Furthermore, correction for amplifier gain is also desired, since experimental runs may be acquired at various gains. Because it is not practical to perform calibration runs for all gain combinations, the compensation file should work with experimental runs obtained at any gain setting. Correction for gain can be achieved by multiplying the fluorescent calibration curves by the ratio of the run gain to the calibration gain for each channel.
Thus, one aspect of this invention is directed toward a method for determining the presence of at least two analytes using at least two fluorescent detecting entities specific for their respective analytes. The fluorescent entities are excited with light having the appropriate wavelength, the fluorescence is determined in at least two spectral channels, and the fluorescent values are corrected for spectral overlap and for temperature dependence of the fluorescent values.
In another embodiment of this invention, the analytes are nucleic acid loci, and the fluorescent entities are specific for their respective loci. The fluorescent entities are excited with light having the appropriate wavelength, fluorescence is measured throughout a range of temperatures, and the signal values are compensated for temperature dependence. In a preferred embodiment, the fluorescent entities are fixed to oligonucleotides having sequences complementary to their respective loci, the oligonucleotides are annealed to the loci, and the measuring step includes monitoring the fluorescence during heating to melt the oligonucleotides from their respective loci.
The apolipoprotein ("Apo E") gene serves as a model system for the present invention. Single base alterations within codons 112 and 158 of the Apo E gene account for the three common alleles (.epsilon.2, .epsilon.3, and .epsilon.4) and six phenotypes of Apo E. Mahley, R. W. (1988) Science 240,622-630. Oligonucleotide targets were synthesized to provide for adjacent hybridization probe genotyping of Apo E alleles. By using artificial templates, the effects of target concentration, complementary strand competition, probe concentration, Mg.sup.++ concentration, and annealing conditions prior to melting curve analysis could be systematically studied irrespective of amplification technique. Thus, an additional embodiment of this invention includes optimizing target concentration and annealing conditions, and reducing complementary strand competition to provide optimal compensated fluorescent measurements.
Additional features of the present invention will become apparent to those skilled in the art upon consideration of the following detailed description of preferred embodiments exemplifying the best mode of carrying out the invention as presently perceived.